Genomic stability depends on faithful replication, accurate repair, and precise inheritance. The maintenance of an intact mitochondrial genome is of critical importance for viability or competitiveness of virtually all eucaryotic cells. In humans, mutations of tRNA, rRNA, and structural genes present in mitochondrial genomes result in severe genetic diseases. In the yeast Saccharomyces cerevisiae, numerous nuclear genetic loci are important for the maintenance of an intact mitochondrial genome. The research proposed here will utilize the unparalleled genetic and biochemical tools of S. cerevisiae to define parameters of mitochondrial genome stability. We have identified genes that encode mitochondrial membrane proteins that are required for the efficient maintenance and inheritance of mtDNA. These proteins reside in both the inner and outer mitochondrial membranes and have discernable activities in three compartments: the cytoplasm (morphology determinants/cytoskeletal attachment), the inter-mitochondrial membrane space (proteolysis assembly factor), and the mitochondrial matrix (DNA binding). Recent work has placed these proteins in a large complex that spans both mitochondrial membranes and associates with mitochondrial DNA, forming a membrane associated nucleoid complex. Our goal is to genetically and biochemically characterize this membrane complex and define its essential role in the maintenance of the mitochondrial genome. Specifically, we propose to: 1) Biochemically characterize the membrane associated mitochondrial nucleoid complex. Native gel electrophoresis, sucrose gradient and gel filtration fractionation, co-immunoprecipitation, and in vivo co-localization assays will be used to define components, detail changes in mutant backgrounds, and probe the roles of specific proteins in the inheritance of the mitochondrial nucleoid complex. 2)Characterize genetic suppressors of mutations affecting the mitochondrial nucleoid complex. Extensive genetic analysis has identified a set of genes encoding both outer and inner mitochondrial membrane proteins necessary for the inheritance of mitochondrial DNA. Suppressors of these loci have been isolated and genetically characterized. These suppressors will be cloned, characterized, and their relationship to the mitochondrial nucleoid complex determined. Taken together, these approaches will further define the genetic and biochemical basis for maintenance of mitochondrial genome integrity.